Phage resistance

ABSTRACT

The present invention relates to the field of dairy science. In particular, the present invention relates to methods for improving dairy starter culture quality as well as food products that can be obtained using such methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit and priority to and is a U.S. National Phase Application of PCT International Application Number PCT/DK2008/050166, filed on Jul. 2, 2008, designating the United States of America and published in the English language, which is an International Application of and claims the benefit of priority to European Patent Application No. 07111640.4, filed on Jul. 3, 2007, and U.S. Provisional Application No. 60/947,759, filed on Jul. 3, 2007. The disclosures of the above-referenced applications are hereby expressly incorporated by reference in their entireties.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of dairy science. In particular the present invention relates to methods for improving dairy starter culture quality.

BACKGROUND OF THE INVENTION

The lactic acid bacterium Lactococcus lactis is used in milk fermentations world wide in the dairy industry to produce a variety of cultured dairy products. Phage infections can ruin the fermentation by inactivating the inoculated cultures. Phages are the major cause of fermentation failures during the manufacture of these cultured dairy products. There is thus a permanent need in the art for L. lactis starter cultures to perform at a high level of consistency and efficiency.

Phages

Lactococcal phages are characterized by having relatively short latent periods and relatively large burst sizes. They are the major cause of fermentation failure leading to production loss in the dairy industry. Lactococcal phages are currently divided into eight distinct groups of which three groups namely “936”, “c2” and “P335” are responsible for the vast majority of phage attacks in industrial fermentations. The genomes of the phages within one single group are highly conserved except for the P335 group.

Industrial fermentations are carried out in large fermentation vats in a non-sterile environment. Prior to fermentation, the ingredients are usually pasteurized. However, the phages are often resistant to the pasteurization process. Presence of phages can lead to variations in flavor and texture of the fermented dairy product or even loss of the entire production with serious economical loss as a consequence. The dairy industry is therefore using a variety of methods in limiting phage attacks. Such approaches include e.g. improved disinfection processes, rotation of starter cultures and application of phage resistant starter strains.

Phage Defense Mechanisms

During evolution L. lactis has developed a series of defense mechanisms against phage attacks. These naturally occurring phage resistance mechanisms (φrm) has been studied extensively and also applied in industrial starter cultures. Most of the naturally occurring φrms are found on plasmids and they are classified into four groups according to their mode of action: 1) adsorption inhibition, 2) blocking of phage DNA injection, 3) restriction/modification systems (R/M) and 4) abortive infection mechanisms (Abi). Among these defense mechanisms, the Abi systems are considered to be the most powerful due to their diverse mode of action and efficiency against the most common phages.

Abi Mechanisms

Abi mechanisms function in the phage life cycle subsequent to the injection of phage DNA into the bacterial cell—typically after expression of early phage genes. As a consequence, the phage lytic cycle is terminated and usually the host dies. Very few viable phage progeny are thus released and the phenotypic outcome is a reduction in the number and size of plaques and thus a reduction of the severity of the phage infection.

To date, twenty-two lactococcal Abi systems have been isolated. These Abi systems target one, two or all three groups of the common phage species 936, c2, P335 with varying efficiency (EOP values from 10⁻¹ to <10⁻⁸) (FIG. 1).

Most of the isolated Abi systems are found on plasmids of which many are conjugative. By sharing the φrms within the bacterial population, conjugation thus provides an adaptation strategy to the phage containing dairy environment. Only a few abi mechanisms have been isolated from the chromosome of L. lactis. This may partly be due to the fact that it is generally easier to isolate genes present on plasmids compared to isolation of genes present on chromosomes. The procedure used in the present invention to isolate a φrm from the chromosome of L. lactis can be used to identify other φrms on the bacterial chromosome.

By isolating spontaneous phage resistant mutants with a similar phenotype with regards to efficiency against a range of phage species it is probably possible to identify strains expressing the abi without having to use genetic modification. Using this method, non-GMO phage resistant strains can thus be isolated. Use of non-GMO starter cultures may be an advantage in some case, in particular in relation to the fact that the legislation in some countries does not allow use of GMO. Furthermore, some consumers tend to prefer non-GMO derived products.

The point of interference with the phage life cycle has been determined to some degree for most of the Abi mechanisms:

-   -   AbiA, AbiF, AbiK, AbiP, AbiR, and AbiT apparently interfere with         phage DNA replication.     -   AbiC apparently interfere with capsid production.     -   AbiE, AbiI, and AbiQ apparently interfere with phage packaging.     -   AbiB is apparently an RNase.     -   AbiD1 seems to interfere with a phage RuvC-like endonuclease.     -   AbiU apparently delays phage transcription.     -   AbiZ apparently causes premature lysis of the infected cell.

These very diverse modes of action are most likely the reason for the very low degree of protein homology that exists between the different Abi mechanisms.

Though the point of action in the phage life cycle has been determined, the phage protein interacting with the Abi mechanism has only been identified in AbiA, AbiD1, AbiK and AbiP. An increasing number of phage genomes are being sequenced providing a bulk of sequence data in which numerous putative proteins are found. However, experimental evidence for the function of these proteins are lacking behind.

Several phage resistant strains of L. lactis have been constructed by introducing abi systems in phage sensitive industrial starter cultures. However, extensive use of these bacterial cultures leads to problems with emergence of phage mutants capable of overcoming the introduced abi systems.

The evolutionary “arms race” between phage mutants and bacterial φrms means that there is a constant need in the art for identifying novel natural φrms. There is a particular need in the art for finding novel Abi-mechanisms that interact with previously unknown targets in the phage. Furthermore there is a need in the art for novel Abi-mechanisms in Lactococcus bacteria that do not classify as GMO. Finally there is a need in the art for identifying φrms that provide efficient protection against phages.

SUMMARY OF THE INVENTION

The present invention thus relates to a polynucleotide conferring at least one phage resistance mechanism to a Lactococcus bacterium, wherein said polynucleotide encodes a polypeptide according to SEQ ID NO 1 and/or SEQ ID NO 2, and/or SEQ ID NO7 or a fragment or variant thereof. The present invention furthermore relates to the polypeptides, uses thereof, expression vectors and cells expressing these polypeptide sequences. The present invention also relates to methods for producing fermented dairy products as well as the products resulting from these processes.

The present invention finally relates to methods for identifying φrms on chromosomal DNA.

The novel φrm(-s) according to the present invention provide a number of advantages as described in the following.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Characteristics of the twenty-two (including AbiV from the present invention) Abi mechanisms isolated to date.

FIG. 2: The sequence from GenBank (acc.nr AF324839) containing orf1 which surprisingly turned out to have the capability to function as a φrm according to the present invention. FIG. 2A: the strains with the transposon containing vector pGhost9::ISS1 inserted on the chromosome. Arrows indicate the position and direction of the inserted ISS1 sequences. The presence of a promoter and the φrm⁺ phenotype is indicated to the right. FIG. 2B: the strains with the cloned fragment including orf1. The lines represent the cloned DNA fragment, and the x in JH-24 represent the position of the frame shift mutation introduced into this strain.

FIG. 3: Time course experiment of a phage infection. Samples are taken during infection of phage resistant Lactococcus lactis strain JH-20 (upper panel) and phage sensitive Lactococcus lactis strain JH-16 (lower panel) with p2 phage. The experiment was run for 120 min and samples were taken at: −10, 0, 10, 20, 30, 40, 50, 60, 90 and 120 minutes. Total DNA was isolated from the cells and restricted with EcoRI. The resulting restriction fragments are representing EcoRI digested p2 DNA. Band 1.3 kb and 4 kb are spanning the cos site which marks the extremities of the phage DNA. The cos site is cut during packaging of phage DNA in the lytic life cycle of the wt phage, revealing mature phage DNA molecules in units of one genome. In the phage resistant Abi mutant, the cos site is not cut resulting in non-mature phage DNA that can not be packed into the phage capsids. The figure thus shows that production of mature phage DNA is significantly decreased in the strains containing the AbiV mechanism.

FIG. 4: DNA sequence of the 1.3 kb DNA fragment (bp 1021-2320 in GenBank acc.nr AF324839) cloned in vector pJH2. This fragment comprises orf1 (bp 1276-1878) encoding the φrm (SEQ ID NO: 1). Ribosome binding site is underlined in nucleotides matching the lactococcal consensus sequence (AGAAAGGAGGT) SEQ ID NO: 19. The translated amino acids are shown below the DNA sequence.

FIG. 5: DNA sequence of the 499 bp DNA fragment from phage p2 containing orf26 and the upstream region towards orf27. Ribosome binding site is underlined in nucleotides matching the lactococcal consensus sequence (AGAAAGGAGGT) SEQ ID NO: 19. The translated amino acids are shown below the DNA sequence (SEQ ID NO: 2).

FIG. 6: Reverse transcriptase PCR carried out on isolated RNA. (A) Experiment done with reverse transcriptase enzyme. (B) Control without reverse transrciptase. Lanes 1-4 represents: JH-80 (spontaneous mutant), JH-20 (high expression of AbiV), JH-54 (wt), JH-32 (insertional mutant), respectively. L is Generuler ladder (Fermentas). The present invention will now be described in more detail in the following.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

Phages: A bacteriophage (from ‘bacteria’ and Greek phagein, ‘to eat’) is any one of a number of virus-like agents that infect bacteria. The term is commonly used in its shortened form, phage. Typically, bacteriophages consist of an outer protein shell (called capsid or head) enclosing genetic material. The genetic material can be ssRNA, dsRNA, ssDNA, or dsDNA between 5 and 500 kilo base pairs long with either circular or linear arrangement. Bacteriophages are much smaller than the bacteria they destroy—usually between 20 and 200 nm in size. Phages according to the present invention have the ability to infect bacteria of the genus Lactococcus.

Phage resistance mechanism: A functional phage resistance mechanism is herein meant to be a mechanism that directly inhibits the phage lytic life cycle. However, phage resistance mechanisms as used herein furthermore denote mechanisms that works in synergy with a phage encoded product. As an example hereof, the present invention relates to use of SEQ ID NO 1 for conferring phage resistance to bacterial cells as well as the use of SEQ ID NO 1 in combination with SEQ ID NO 2 for obtaining an even more efficient phage resistance mechanism than was possible when only using SEQ ID NO 1.

Lactococcus: is a lactic acid bacterial genus of five major species formerly included as members of the genus Streptococcus Group N and related species. They are gram-positive bacteria, and they are typically spherical or ovoid, 0.5-1.2 μm by 0.5-1.5 μm, and occur in pairs and short chains. They are non-spore forming and are not motile. The type species for the genus is L. lactis which in addition have two subspecies lactis and cremoris. Lactococcus is commonly used in the dairy industry in the manufacture of fermented dairy products. They can be used in single strain starter cultures, or in mixed strain cultures comprising other strains of Lactococcus or lactic acid bacteria such as e.g. Leuconostoc, Lactobacillus and Streptococcus.

A fragment: A fragment according to the present invention is herein defined as a fragment of a polypeptide being at least 100 amino acids, preferably at least 110, more preferably at least 120 amino acids. With regards to SEQ ID NO 1, the fragment is preferably at least 100 amino acids in length, more preferably at least 125 amino acids in length, more preferably at least 150 amino acids in length, more preferably at least 175 and most preferably at least 190 amino acids in length.

Promoter: The term “promoter”, “promoter region” or “promoter sequence” refer generally to transcriptional regulatory regions of a gene, which may be found at the 5′ or 3′ side of the coding region, or within the coding region, or within introns. As used herein the term promoter shall include any portion of genomic DNA (including genomic DNA disclosed herein), which is capable of initiating expression of but not limited to operably linked nucleotide sequences at levels detectable above background. In the context with the present invention a “strong promoter” shall be understood as a promoter which results in expression of a polypeptide according to the invention, wherein the level of expression is significantly higher compared to the endogenous homologous promoter in the Lactococcus genome. The level of expression can be detected and/or measured by e.g. Northern blot, real-time PCR, reporter gene assays, etc.

Expression vector: A vector is a component or composition for facilitating cell transduction or transfection by a selected nucleic acid, or expression of the nucleic acid in the cell. Vectors include, e.g., plasmids, cosmids, viruses, BACs, PACs, P1, YACs, bacteria, poly-lysine, as well as linear nucleotide fragments etc. An “expression vector” is a nucleic acid construct or sequence, generated recombinantly or synthetically, with a series of specific nucleic acid elements that permit transcription of a particular nucleic acid sequence in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. The expression vector typically includes a nucleic acid to be transcribed operably linked to a promoter. The nucleic acid to be transcribed is typically under the direction or control of the promoter. The expression vector may replicate autonomously in the host cell or may integrate into the host genome after the transfection or transduction and replicate as part of the genome. Finally “an expression vector” encoding more than one polypeptide sequences according to the present invention comprises the situation wherein one expression vector comprises polynucleotide sequences encoding more than one polypeptide product as well as the situation wherein the polynucleotide sequences are cloned into two different expression vectors.

pGhost9::ISS1: The term “pGhost9::ISS1” covers a vector with an antibiotic resistance marker, a Lactococcus replicon, and preferably also an E. coli replicon. The replicon is thermosensitive allowing for selection for integration into the host chromosome. Also the vector contains an insertion sequence that enables random integration of the vector into the host chromosome. It follows that vectors with similar functions may be used in connection with the present invention.

Identity: The term “identity” or “sequence identity” is a measure of the degree of identity between polynucleotide sequences on a nucleotide-by-nucleotide basis or amino acid-by-amino acid basis, respectively over a window of comparison. Sequences according to the present invention have an identity of at least 70% to SEQ ID NO 1, or a fragment thereof.

Food products: Food products according to the present invention include milk based products that have been subject to fermentation processes. Examples thereof include: sour cream, crème fraîche, buttermilk, butter, cheese, cottage cheese, quark, cream cheese, fromage frais, yoghurt, etc. However, other types of food products may also be produced using fermentation or fermentative microorganisms according to the present invention such as e.g. fruit juices, fermented vegetables/fruits, processed meat products, etc.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

In a first aspect the present invention thus relates to an isolated polynucleotide sequence that encodes a polypeptide with at least 70% identity, preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 70% identity, preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, and most preferably at least 95% identity with SEQ ID NO 1 (AbiV from Lactococcus lactis), or a fragment thereof, and wherein expression of said polynucleotide confers at least one phage resistance mechanism to a Lactococcus bacterium. This polynucleotide sequence is found naturally in the Lactococcus bacterium, but it is normally not transcriptionally active. It has surprisingly been found that expression of this polypeptide confers a previously unknown phage resistance mechanism to the bacterium.

A second aspect of the present invention relates to an isolated polynucleotide derived from a Lactococcus lactis phage that encodes a polypeptide with at least 70% identity, preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 95% identity, preferably at least 97% identity, and most preferably at least 99% identity with SEQ ID NO 2, or a fragment thereof and/or an isolated polynucleotide that encodes a polypeptide with at least 70% identity, preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 95% identity, preferably at least 97% identity, and most preferably at least 99% identity with SEQ ID NO 7. Optionally, the polynucleotide sequence may encode at least one of SEQ ID NO 1, SEQ ID NO 2, and SEQ ID NO 7 or any variant thereof in the form of one or more polynucleotide sequences.

SEQ ID NO 2 and SEQ ID NO 7 are phage proteins. The inventors have found out that these proteins most likely need to be mutated in order for the phage to escape the phage resistance mechanism conferred by expression of SEQ ID NO 1 or variants thereof. Phage proteins according to the present invention therefore have at least 70% identity with SEQ ID NO 2 and/or SEQ ID NO 7 in order to provide functional phage protein that may suppress the effects of emergence of mutated phage protein that could potentially suppress the effects of the translated SEQ ID NO 1 protein or variants thereof. In a preferred embodiment according to the present invention, polynucleotide sequences encoding both SEQ ID NO 1 or variants thereof as well as SEQ ID NO 2 and/or SEQ ID NO 7 or variants thereof, are thus provided thus conferring highly efficient phage protection mechanisms to a host cell. It furthermore follows that the invention relates to expression vectors as well as Lactococcus bacteria and/or starter cultures comprising polynucleotide sequences encoding such polypeptide sequences.

In a third aspect, the present invention relates to an isolated polypeptide conferring at least one phage resistance mechanism to a Lactococcus bacterium, wherein said polypeptide is selected from one or more of the group consisting of: a polypeptide with at least 70% identity with SEQ ID NO 1, or a fragment thereof, a polypeptide with at least 70% identity with SEQ ID NO 2, or a fragment thereof, and a polypeptide with at least 70% identity with SEQ ID NO 7, or a fragment thereof.

A fourth aspect relates to the use of one or more polynucleotides according to the present invention and/or one or more polypeptides according to the present invention for improving phage resistance in a Lactococcus bacterium.

A fifth aspect relates to a method for fermenting food product, said method comprising the step of adding one or more of the components according to the present invention. The invention furthermore relates to products that can be obtained and/or are obtained using this method.

A sixth aspect relates to a method for obtaining phage resistant bacterial cells, said method comprising use of pGhost9::ISS1 (or similar systems) for random insertion into a bacterial cell and subsequently screening and selecting for phage resistant cells. The invention furthermore relates to cells that can be obtained and/or are obtained by such methods. In a preferred embodiment, the cell is a Lactococcus bacterium wherein a polynucleotide encoding SEQ ID NO 1 (or a variant thereof) is transcriptionally active.

A final aspect relates to a Lactococcus bacterium that expresses at least one polypeptide selected from the group consisting of: a polypeptide with at least 70% identity with SEQ ID NO 1, or a fragment thereof, a polypeptide with at least 70% identity with SEQ ID NO 2, or a fragment thereof, and a polypeptide with at least 70% identity with SEQ ID NO 7, or a fragment thereof.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the following non-limiting examples.

EXAMPLES Example 1

Bacterial Strains, Plasmids, and Media

Strains and plasmids used in this invention are listed in table 1. Escherichia coli was grown at 37° C. in LB medium. Lactococcus lactis was grown in M17 with the supplement of 0.5% glucose (GM17). Lactococci were grown at 30° C. except strains containing the thermo sensitive vector pGhost9::ISS1. These strains were grown at 28° C. for replication of the vector or 36° C. to avoid replication. When appropriate, antibiotics were added as follows: E. coli, 100 μg/ml of ampicillin, 10 μg/ml of chloramphenicol, 150 μg/ml of erythromycin; for L. lactis, 5 μg/ml of chloramphenicol, 3 μg/ml of erythromycin.

TABLE 1 List of bacteria, phages and plasmids used in the invention Bacterial strain, phage or plasmid Characteristic Source Lactococcus lactis SMQ-86 Lactococcus lactis subsp. cremoris. Multiple plasmids, pSA3, host for the tested P335 phages. Erm^(R) (2) IL1403 Lactococcus lactis subsp. lactis IL1403, host for some 936 phages (1) MB112 Lactococcus lactis subsp. cremoris MG1363, Δupp, Host for 936 and c2 phages (7) JH-20 MB112 (pJH2); Cam^(R), Abi⁺ This Invention JH-22 IL1403 (pJH2); Cam^(R), Abi⁺ This Invention JH-23 SMQ-86 (pJH2); Cam^(R), Abi⁺ This Invention JH-24 MB112 (pJH3); Cam^(R), Abi⁻ This Invention JH-25 MB112 (pJH4); Cam^(R), Abi⁺ This Invention JH-26 MB112 (pJH5); Cam^(R), Abi⁺ This Invention JH-32 MB112 (pGhost9::ISS1 inserted in Lin at bp1962); Erm^(R), grown at 36° C., Abi⁺ This Invention JH-46 MB112 (pGhost9::ISS1 inserted in Lin at bp1962); Erm^(R), grown at 36° C., Abi⁺ This Invention JH-47 MB112 (pGhost9::ISS1 inserted in Lin at bp2296); Erm^(R), grown at 36° C., Abi⁺ This Invention JH-48 MB112 (pGhost9::ISS1 inserted in Lin at bp2240); Erm^(R), grown at 36° C., Abi⁺ This Invention JH-49 JH-32 cured for pGhost9, leaving ISS1 in place; Abi⁻ This Invention JH-50 JH-46 cured for pGhost9, leaving ISS1 in place; Abi⁻ This Invention JH-51 JH-47 cured for pGhost9, leaving ISS1 in place; Abi⁻ This Invention JH-52 JH-48 cured for pGhost9, leaving ISS1 in place; Abi⁻ This Invention JH-53 MB112 (pJH6); Cam^(R), Abi⁻ This Invention JH-54 MB112 (pLC5); Cam^(R), Abi⁻ This Invention JH-80 MB112 (spontaneous mutation to express φrm); Abi⁺ This Invention JH-81 transconjugant with transferred φ rm and Erm^(R) from JH-32 to LKH208; Abi⁺, Erm^(R), Rif^(R), Strep^(R), FU^(S) This Invention MG1614 MG 1363 Rif^(R), Strep^(R) (M. Gasson) (5) Escherichia coli EC1000 RepA⁺ MC1000, Km^(R) (4) JH-56 EC1000 (pJH7), Erm^(R) This Invention JH-57 EC1000 (pJH8), Erm^(R) This Invention JH-58 EC1000 (pJH9), Erm^(R) This Invention JH-59 EC1000 (pJH10), Erm^(R) This Invention TOP10F′ Chemically competent cells from the TOPO TA cloning kit Invitrogen JH-19d EC1000 (pJH6), Cam^(R) This Invention phages p2 Small isometric headed, 936 species S. M sk1 Small isometric headed, 936 species F. V jj50 Small isometric headed, 936 species F. V 712 Small isometric headed, 936 species S. M P008 Small isometric headed, 936 species S. M bIL170 Small isometric headed, 936 species S. M c2 Prolate headed, c2 species S. M bIL67 Prolate headed, c2 species S. M ml3 Prolate headed, c2 species S. M eb1 Prolate headed, c2 species S. M ul36 Small isometric headed, P335 species S. M KITI Small isometric headed, P335 species S. M Ø31 Small isometric headed, P335 species S. M Ø50 Small isometric headed, P335 species S. M Q33 Small isometric headed, P335 species S. M Q30 Small isometric headed, P335 species S. M P335 Small isometric headed, P335 species S. M p2.1 Small isometric headed, 936 species, deletion in orf26 This Invention sk1.1 Small isometric headed, 936 species, nonsense mutation in orf26 This Invention jj50.1 Small isometric headed, 936 species, nonsense mutation in orf25 (homologue to p2 orf26) This Invention P008.1 Small isometric headed, 936 species, nonsense mutation in orf33 (homologue to p2 orf26) This Invention bIL170.1 Small isometric headed, 936 species, nonsense mutation in e24 (homologue to p2 orf26) This Invention c2.1 Prolate headed, c2 species, mutation in e11 (homologue to p2 orf26) This Invention plasmids pCI372 Shuttle vector for E. coli and L. lactis. No promoter in front of multiple cloning site; Cam^(R) (3) pLC5 Expression vector for L. lactis and E. coli. Promoter in front of PstI site used for cloning; Cam^(R) This Invention pGhost9::ISS1 pGhost9::ISS1, temperature sensitive vector with insertion sequence used for random mutagenesis, (6) Erm^(R) pJH2 bp 1021-2320 on Lin sequence* cloned in PstI site of pLC5; Cam^(R) This Invention pJH3 pJH2, restricted in ClaI site of AbiV and filled with Klenow, gives frameshift mutation; Cam^(R) This Invention pJH4 pJH2 isolated from JH22 This Invention pJH5 pJH2 isolated from JH23 This Invention pJH6 bp 1021-2320 on Lin sequence* cloned in PstI and XbaI sites of pCI372 This Invention pJH7 HindIII rescue of pGhost9::ISS1 with flanking chromosomal DNA from JH-32 This Invention pJH8 HindIII rescue of pGhost9::ISS1 with flanking chromosomal DNA from JH-46 This Invention pJH9 HindIII rescue of pGhost9::ISS1 with flanking chromosomal DNA from JH-47 This Invention Lin sequence refers to GeneBank acc nr AF324839 Cam^(R), chloramphenicol resistance; Amp^(R), ampicillin resistance; Erm^(R), Erythromycin resistance; Km^(R), Kanamycin resistance; Rif^(R), Rifampicin resistance; Strep^(R), Streptomycin resistance; FU^(R) fluorouracil resistance Abi⁺, phage resistance phenotype; Ab⁻, phage sensitive phenotype F. V = Finn K. Vogensen, University of Copenhagen S. M = Sylvain Moineau

Example 2

Bacteriophage Propagation and Assays

Bacteriophages used in this invention are listed in table 1. Bacteriophages sk1 and jj50 were kindly provided by F. K. Vogensen (University of Copenhagen). Prior to use all phages were purified two times by picking a single plaque with a sterile Pasteur pipette and plating it on a sensitive host. Propagation of phages to obtain high titer lysates was performed in two steps:

In the first propagation a single plaque was transferred into a fresh ON culture of a sensitive host inoculated (1%) in GM17 supplemented with 10 mM CaCl₂ and incubated at 30° C. (or 36° C. in the case of pGhost9::ISS1 containing host strains) until lysis. The lysate was filtered through a 0.45 μm syringe filter.

The second propagation was performed by inoculating an exponentially growing host culture at OD₆₀₀=0.2 with phages from the first propagation (10⁴ pfu/ml) in GM17+10 mM CaCl₂.

The culture was then incubated with agitation (200 rpm) until lysis at the same temperature as for the first propagation. The lysate was filtered (0.45 μm filter). The titer of phage lysates was determined using conventional methods.

Efficiency of plaguing (EOP) was calculated by dividing the titer on the tested strain with the titer on the sensitive wt strain. Adsorption assays were conducted as described by Sanders and Klaenhammer (17) except a 5 min incubation period was used instead of 15 min. Cell survival was assayed by the method of Behnke and Malke (2) using a multiplicity of infection (MOI) of 5. One-step growth assay (and determination of burst size) and center of infection (COI) assay was performed as described previously (14) by using MOIs of 0.2 and 0.5, respectively. ECOI (efficiency of COI) was determined by dividing the number of COI from the resistant strain by the number of COI from the sensitive strain. Replication of phage DNA was followed in a time course experiment using the method of Hill et al. (8). Visualization of phage DNA by labeling with the fluorescent dye SYBR-Gold was performed as described by Noble and Fuhrman (15) with the following modifications: The original SYBR-Gold solution was diluted (×1000). Phage lysate to be stained was treated with 1 μg/ml DNAse and RNAse and incubated for 30 min at 37° C. The lysate was stained with the diluted SYBR-Gold to give 2.5% final concentration (vol/vol) diluted SYBR-Gold and left ON at 4° C. in the dark. One μl of the labeled phage stock was mixed with 1 μl exponentially growing cell culture and visualized under a Zeiss axioplan epifluorescence microscope.

Example 3

Mutagenesis with pGhost9::ISS1

Random integration of the vector pGhost9::ISS1 into the chromosome of MB112 and subsequent cloning of flanking chromosomal DNA was performed essentially using the method of Maguin et al. (10). The method of Maguin, however, is normally used to identify inactivation of genes by randomly inserting the construct in chromosomal genes, thereby inactivating them. Subsequent selection for a desired phenotype enables screening for strains containing a loss of function mutation. The fact that all inspected mutants in the present invention had insertions in non coding regions or genes upstream of orf1 together with the observation that presence of the complete vector pGhost9::ISS1 was needed for the Abi⁺ phenotype led to the hypothesis that the abiV gene (orf1) was transcribed from the promoter encoding the erythromycin resistance gene in pGhost9::ISS1 (FIG. 2). Previous studies have reported promoter activity in the ISS1 sequence (5). No effect on phage resistance phenotype of such promoter activity was observed in the present invention. It has not previously been shown that random insertion of the vector and subsequent transcription from the promoter of the erythromycin resistance gene can be used to activate existing biological mechanisms, such as e.g. Abi-mechanisms.

To ensure that the mutations in the isolated strains had arisen from independent events, the integration step (growth at 37° C.) was performed on 12 separate cultures. After the integration step, the cultures were diluted ×10.000 in GM17+3 μg/ml Erythromycin and left for phenotypic expression ON at 37° C. These cultures were inoculated (1%) and when growing exponentially aliquots were removed. 10 mM CaCl₂ (final concentration) was added to these aliquots before inoculating with phage sk1 (MOI>1). After 10 min incubation at 37° C. the cultures were spread on selective GM17+Erm plates. A number of phage resistant colonies were isolated and purified from each of the 12 independent cultures. Four strains were chosen from independent cultures to identify the location of the inserted pGhost9::ISS1. This was performed by rescuing of the inserted vector and cloning of flanking chromosomal DNA. The cloned chromosomal DNA fragments were subsequently sequenced

Example 4

DNA Isolation and Manipulation

Plasmid DNA was isolated from E. coli and L. lactis using the QIAprep Spin Miniprep Kit (Qiagen); for L. lactis however, lysozyme (15 mg/ml) was added to buffer p1 and the solution with the resuspended cells was incubated at 37° C. for 30 min before proceeding with the manufacturers protocol. Phage DNA was prepared using the Qiagen Lambda Maxi Kit (Qiagen) with the addition of proteinase K (20 mg/ml) to buffer L3 and subsequent incubation at 65° C. for 30 min before adding buffer L4. Total intracellular DNA was isolated using the method of Hill et al. (8). Restriction enzymes, T4 DNA ligase and Klenow fragment (Fermentas) were used according to the manufacturer's instructions. Electroporation of E. coli and L. lactis was performed as described previously (13). The DNA fragment corresponding to bp 1021 to 2320 (FIG. 4) in the GenBank sequence AF324839 was subcloned in the TOPO TA cloning kit prior to cloning in pCI372 and pLC5.

Example 5

DNA Sequencing and Sequence Analysis of DNA and Protein

Oligonucleotide sequences used for plasmid constructions and sequencing: For sequencing the flanking chromosomal DNA of the rescued pGhost9::ISS1 inserts a primer located in the ISS1 was used (5′-GAAGAAATGGAACGCTC-3′). Phage genome sequencing was performed with an ABI prism 3700 apparatus from the genomic platform at the research center of the Centre Hospitalier de I'Université Laval using a set of oligonucleotides previously used for sequencing of 936 phage genomes (11).

Sequence data was assembled using the Staden Pregap4 version 1.5. Sequence homology searches in databases were done using BLAST (1). Molecular weight and pI of the investigated proteins were estimated using the Protein Calculator at the website: http://www.scripps.edu/˜cdputnam/protcalc.html

Example 6

A Phage Resistance Mechanism (φrm) is Found on the Chromosome of Lactococcus lactis subsp. cremoris MG1363

L. lactis subsp. cremoris MG1363 is sensitive to infection of phages from the 936 and c2 species. In this invention a transposon mutagenesis system (described in details in (10)) was used to identify a novel φrm on the chromosome of MG1363. The system (pGhost::ISS1) comprises the vector pGhost9 containing an erythromycin resistance gene (Em^(r)) and the ISS1 insertion sequence which allows for random integration of the construct into the host chromosome. Due to a thermosensitive origin of replication (plasmid is not replicating at 37° C.) it is possible to select for mutants with the construct inserted in the chromosome by growing at 37° C. in the presence of erythromycin, allowing for phenotypic expression by growing at selective conditions ON.

This selection was done for a number of independently grown cultures resulting in isolation of independent integration events. These cultures were screened for resistance to phage sk1 by selecting colonies growing on erythromycin plates in the presence of phages (MOI>1). The frequency of mutations conferring phage resistance was 100 times higher in cultures with mutants containing the pGhost::ISS1 inserts compared to the control cultures in which the phage resistance was caused by spontaneous mutations. This clearly indicates that the mutations in the phage resistant mutants containing pGhost9::ISS1 in most cases were caused by the insertion of this construct.

A number of Em^(r)/φrm⁺ colonies were isolated. From four of these independently mutagenized cultures, the inserted construct was obtained along with a piece of flanking chromosomal DNA. Sequence analysis revealed insertions on the chromosome corresponding to bp 1962 (strains JH-32 and JH-46), bp 2240 (JH-48) and bp 2296 (JH-47) on the sequence available in GenBank under the accession number AF324839 (hereafter designated Lin). Bp 1021 to bp2320 therein corresponds to SEQ ID NO 6. All strains had insertions in the intergenic region between two genes (designated orf1 and trans) or in the 3′ end of the trans gene (FIG. 2A). There are no genes in the same orientation immediately downstream of orf1 and since the mutagenizing constructs were all inserted in the same orientation pointing towards orf1 it was hypothesized that orf1 is encoding a φrm which is transcribed from the Em^(r) gene promoter when pGhost::ISS1 is inserted upstream of orf1. Curing the strains for the vector (leaving a single copy of ISS1 at the insertion site) resulted in φrm⁻ phenotype supporting the hypothesis that a promoter in pGhost::ISS1 is needed for transcription of orf1 and the resulting φrm⁺ phenotype. This implies that orf1 is silent in wt MG1363 which is supported by the phage sensitive phenotype of this strain.

Example 7

Identification of orf1 as a φrm

To test if orf1 is a φrm, a fragment corresponding to bp 1021 to 2320 on the Lin sequence was cloned in the shuttle vector pCI372 (pJH6) and in the expression vector pLC5 (pJH2). These constructs were transformed in MB112 and the resulting strains (JH-53 and JH-20, respectively) were tested by cross streaking assay for resistance to phage p2. JH-53 containing pJH6 with no promoter upstream of orf1 showed no phage resistance phenotype. In comparison, JH-20 containing pJH2 with orf1 cloned downstream of a strong promoter revealed phage resistance phenotype.

To verify orf1 as being the φrm, a frameshift mutation was introduced in orf1 by filling a unique ClaI site with Klenow fragment followed by ligation and transformation of this vector (pJH3) in wt MB112. The mutated orf1 was sequenced verifying the frameshift mutation. The resulting strain JH-24 had a phage sensitive phenotype and it was therefore concluded that orf1 is encoding a φrm.

Example 8

The Isolated φrm is Effective Against Phages of the 936 and c2 Species

The three phage species 936, c2 and P335, known to be responsible for the majority of phage caused fermentation failures were tested for their sensitivity to the φrm. Four strains of the 936 species were tested against JH-20. Efficiency of plaquing (EOP) values around 10⁻⁴ were obtained for phages p2, sk1 and jj50 while phage 712 was insensitive to the φrm (Table 2). pJH2 was inserted into the host JH-22 (L. lactis subsp. lactis IL1403) which is sensitive to the 936 phages P008 and bIL170. When tested against these phages the φrm revealed EOP values around 10⁻⁴. Similar values were obtained when testing JH-20 against four phages of the c2 species (Table 2). Similar EOP values were obtained for MB112 and JH-54 when tested against the 936 and c2 phage species (data not shown), thus ruling out the possibility for the vector pLC5 being responsible for the φrm⁺ phenotype.

TABLE 2 Phage Host strain EOP 936 species^(a) sk1 JH-20 2.7 ± 1.4 × 10⁻⁴ p2 JH-20 4.8 ± 1.8 × 10⁻⁴ jj50 JH-20 8.3 ± 0.5 × 10⁻⁵ 712 JH-20 1.1 ± 0.2 P008 JH-22 3.8 ± 1.5 × 10⁻⁴ bIL170 JH-22 3.1 ± 1.2 × 10⁻⁴ c2 species^(a) c2 JH-20 5.2 ± 0.4 × 10⁻⁵ bIL67 JH-20 2.0 ± 1.2 × 10⁻⁴ ml3 JH-20 3.4 ± 0.3 × 10⁻⁴ eb1 JH-20 2.2 ± 0.7 × 10⁻⁴ P335 species^(b) ul36 JH-23 1.0 KITI JH-23 1.6 Ø31 JH-23 1.0 Ø50 JH-23 1.0 Q33 JH-23 0.7 Q30 JH-23 0.8 P335 JH-23 0.4 ^(a)EOP of 936 and c2 species is 1.0 on both L. lactis subsp. cremoris MG1363 (MB112) and MB112 + pLC5 (JH-54). EOP of phages P008 and bIL170 is 1.0 on L. lactis subsp. lactis IL1403 ^(b)EOP of P335 species is 1.0 on L. lactis subsp. cremoris (SMQ-86).

To test the φrm for efficiency against P335 phages, the φrm was inserted in a suitable host (SMQ-86) resulting in the strain JH-23. When tested against seven species of P335 phages EOP values around 1 were obtained. To rule out the possibility that modifications had taken place rendering the φrm inefficient, pJH2 was prepared from JH-23 and re-inserted into MB112 to give strain JH-26. Tests against phage p2 showed an intact φrm phenotype.

Those results showed that the φrm found on the chromosome of L. lactis subsp. cremoris MG1363 and expressed from pJH2 is effective against phages from most of the tested 936 species and all tested c2 species while no effect was seen on P335 species.

The results also showed that the φrm encoded by orf1 is efficient in both the subspecies (cremoris and lactis) of L. lactis.

Furthermore the results showed that EOP values did not vary whether the φrm was expressed from a promoter located in single copy on the chromosome of the host or from a strong promoter on the vector pJH2. This indicates that the efficiency of the system is not dependent on the copy number of the gene.

Example 9

Temperature Sensitivity

The efficiency of the φrm was tested against phage sk1 at 30° C. and 37° C. EOP values were in both cases around 10⁻⁴ (data not shown) indicating that the φrm is stable within this temperature range.

Example 10

Type of Phage Resistance Mechanism

A series of microbiological experiments were conducted to determine the type of φrm encoded by orf1.

An adsorption assay showed that the level of adsorption of phage p2 to cells with the expressed φrm was 95.9±10.6% compared to wt MG1363 (data not shown).

An assay was conducted where the φrm⁺ strains JH-32, JH-46, JH-47, JH-48 and control MB112 was infected with sk1 that had been fluorescently labeled with the DNA binding dye SYBR-Gold. Following infection the fluorescently labeled phage DNA could be visualized under an epifluorescence microscope. Immediately following phage infection (MOI=10) of wt strain MB112 a fluorescent halo of adsorbed phages was seen surrounding the host cells. Less than 10 min after infection the fluorescent signal on the cell surface was decayed and instead a very bright fluorescent signal was observed in the center of the cell, thus indicating that the phage DNA had been injected into the host cell (data not shown).

The same result was obtained with the strains having the φrm⁺ phenotype. This supports the data from the adsorption assay and also shows that the phage DNA is being injected in the φrm containing cells. These results indicate that the φrm is not an adsorption or injection blocking mechanism.

A cell survival assay showed no increased survival on cells harboring the φrm (Table 3) indicating that the host dies upon infection. The plaque size of phage p2 was smaller when assayed on φrm⁺ cells compared to φrm⁻ cells (Table 3). Finally, total DNA extraction from φrm⁺ cells during a time course experiment of infection with phage p2 showed phage DNA replication which persisted in the cell throughout the experiment (FIG. 3).

TABLE 3 Assay MB112 (wt) JH-20 (abiV) EOP^(a) 1.0 4.8 ± 1.8 × 10⁻⁴ ECOI (%)^(b) 1.0  0.5 ± 0.2 Burst size (pfu/cell)^(c) 38.8 ± 5.7 11.1 ± 5.2 fraction surviving cells^(d) 6.1 ± 1.3 × 10⁻⁵ 3.1 ± 0.3 × 10⁻⁶ phage DNA replication^(e) + + (concatemeric) plaque size (mm) 1.5-1.7 pinpoint - 0.7 ^(a)n = 3 ^(b)MOI = 0.5, n = 3 ^(c)MOI = 0.2, n = 3 ^(d)MOI = 5, n = 3 ^(e)MOI = 2, n = 1

All the above results confirm that the mechanism is a φrm that functions as an abortive infection mechanism. This was named AbiV.

Example 11

Sequence Analysis of the 1.3 kb DNA Fragment Containing the φrm

The DNA fragment cloned in pJH2 was sequenced (SEQ ID NO 3 and 5). The fragment consists of 1300 nucleotides. Nucleotides 1 to 1300 correspond to nucleotides 1021 to 2320 in the Lin sequence (GenBank acc.nr: AF324839). One significant open reading frame (orf) was found encoding the polypeptide sequence shown in SEQ ID NO 1. This gene encoding the φrm was named abiV (SEQ ID NO 3) and the translated protein was named AbiV (SEQ ID NO 1). The G+C content of the gene was found to be 31.7%. This value is typical for abi mechanisms which are known to have lower G+C contents compared to the normal 37% in L. lactis. Searches for promoter sequences upstream of abiV (bp 1 to 430) were performed but no suitable promoter could be found in this region. This corresponds well with the hypothesis of the φrm being silent in the wt strain MB112. The translation start codon was preceded (8 bp upstream) by a ribosome binding site (5′-TGAACGGAGAG-3′, underlined sequence matches consensus sequence).

Example 12

Analysis of the AbiV Protein Encoded by abiV in pJH2

Since the abiV gene is the only orf in the cloned sequence of pJH2 and a frame shift mutation in this orf causes the phage sensitive phenotype, it is concluded that the protein encoded by this gene is responsible for the φrm⁺ phenotype. AbiV consists of 201 amino acids and has a molecular weight of 22692 Da. The pI was estimated to be 5.37.

The protein does not contain any putative transmembrane or signal peptide motifs and it is therefore likely that the protein is cytosolic. Homology searches in databases did not reveal any homology (at amino acid or nucleotide level) to other lactococcal proteins or any proteins with known function. Likewise, no conserved domains were found in the protein.

The deduced function of AbiV is therefore new and the φrm is a novel abi mechanism.

Example 13

Effect of AbiV on Phage Life Cycle

The effects of the AbiV system was tested on the phage p2 life cycle using the phage sensitive strain MB112 and the corresponding AbiV containing strain JH-20. The following results are summarized in table 3.

The propagation of p2 on JH-20 was inhibited as seen by the EOP of ca 10⁻⁴ and the plaque size was reduced from about 1.5 mm to <1 mm. Very few of the infected cells harboring the φrm survived infection.

The ECOI on JH-20 was 0.5±0.2% indicating that only 5 out of 1000 infected cells managed to release at least one viable phage. In these successful infections the burst size was reduced by 72% (from 38.8±5.7 in MB112 to 11.1±5.2 in JH-20).

The combined negative effects of AbiV on cell survival, ECOI and burst size were the cause of the reduced plaque size and EOP of p2 on JH-20.

The replication of phage DNA was followed in a 2 h phage infection experiment of p2 on phage resistant JH-20 and phage sensitive MB112 (FIG. 3). Phage DNA was visualized by digesting the total DNA prepared from an infected cell culture with EcoRV and comparing the resulting fragments run on an agarose gel with the EcoRV restriction map of phage p2.

Ten minutes after infection replication of phage DNA was observed in both strains. In MB112 the concentration of phage DNA decreases around 40 min after infection coinciding with lysis of the sensitive host culture. On the contrary, in JH-20 the phage DNA persists throughout the experiment which was terminated after 2 h. Inspecting the EcoRV digested phage DNA fragments, two bands of 1.3 and 4 kb respectively and a 5.3 kb fragment are seen in the phage sensitive culture. The 5.3 kb fragment is spanning the cos site on the phage DNA which is the site where the replicated phage DNA is cut into identical units of complete phage genomes before packaging of the DNA into the capsids. Therefore the 1.3 and 4 kb fragments represent DNA that has been cut at the cos site. The presence of both non-resolved and resolved DNA in the phage sensitive strain is due to the continuous DNA replication throughout the phage life cycle and the simultaneous packaging of already resolved DNA into the phage capsids. In JH-20 (AbiV⁺) only the 5.3 kb fragment is observed which indicates that the phage DNA is not cut at the cos site in this strain.

The above results show that AbiV works after phage DNA replication and is thus categorized as a late abi mechanism. The presence of concatemeric DNA fragments (cos site not cut) further suggests that the φrm might work at a late stage for example during packaging of phage DNA into the capsids.

Example 14

Phage Genes Involved in Sensitivity to AbiV

A number of phage mutants capable of overcoming AbiV were isolated. On JH-20 AbiV-insensitive mutants of p2, sk1, jj50 and c2 were isolated and named p2.1, sk1.1, jj50.1 and c2.1, respectively. On JH-22, mutants of P008 and bIL170 were isolated and named P008.1 and bIL170.1, respectively.

The full genome of mutant p2.1 was sequenced revealing only mutations in the region around the early gene orf26 (SEQ ID NO 4). SEQ ID NO 4 encodes a polypeptide sequence denoted SEQ ID NO 2. The following polynucleotide mutations were found in phage p2.1 that escaped the AbiV-mechanism:

-   -   Two point mutations in orf26 leading to amino acid changes.     -   One point mutation in the intergenic region between orf26 and         the upstream gene orf27     -   A 55 bp deletion including the startcodon and 6 downstream base         pairs of orf26.

The homologues of p2 orf26 in the other phage mutants were sequenced. Nonsense mutations were observed in: orf26 (sk1.1), orf25 (jj50.1), orf33 (P008.1), e24 (bIL170.1) and a point mutation leading to an amino acid change (T to P) was seen in ell (c2.1).

These data show that AbiV-resistant phage mutants apparently fail to produce functional protein encoded by an early gene homologous to phage p2 orf26. In at least one phage mutant (p2.1), orf26 is the only gene which is mutated. Finally, phage 712 (936 species) is the only phage among the tested phages from the 936 and c2 species that does not contain an orf26 homologue. Among the wt phages of the 936 and c2 species, this phage is also the only one which is not sensitive to AbiV.

Based on the above results, it is concluded that a functional copy of phage p2 orf26 (and homologues in other phage species) is mandatory for successful φrm⁺ phenotype of AbiV. The gene is named sav (sensitivity to abiV) and the translated putative protein was named Sav. It is thus possible to strengthen the AbiV-mechanism by supplying the AbiV host cell with a polynucleotide sequence encoding wt Sav.

A nucleotide blastn analysis orf phage p2 orf26 revealed a high degree of sequence homology to other lactococcal phage genes: jj50 orf25 (99.7%), sk1 orf26 (99.0%), P008 orf33 (91.4%), bIL170 e24 (90.6%). Furthermore the translated p2 orf26 showed a more distant relationship (29%) with phage c2 gene ell. Despite the low degree of homology the e11 gene of phage c2 is involved in sensitivity to AbiV since a mutation in this gene helps the phage c2.1 escape AbiV. Therefore, sequences of either phage 936-like orf26 homologues (SEQ ID NO 2) or c2-like e11 homologues (SEQ ID NO 7; DNA sequence: SEQ ID NO 8, derived from accession number NC001706 disclosing the complete genome of Lactococcus lactis phage c2), or variants or fragments thereof are a part of the present invention.

Example 15

Analysis of the Phage p2 Gene orf26 (sav) and the Putative Protein (Sav) Encoded by this Gene

The DNA fragment containing phage p2 gene orf26 and the upstream intergenic region to orf27 was sequenced on both strands. The sequenced fragment contains 499 nucleotides (SEQ ID NO 5). The sav gene consists of 384 bp (SEQ ID NO 4). Upstream of sav in a suitable (8 bp) distance is found a RBS sequence (GGATTGGGGGT, underlined sequence matches consensus sequence). No promoter sequence is found in the region between orf27 and sav. This corresponds well with the genetic structure of this region in p2 and in the closely related phage sk1. In both phages orf26 is the last gene in a putative operon consisting of orf30 to orf26 where the promoter is upstream of orf30 (4).

The sav gene is located at the end of the early transcribed region of phage p2. The putative protein Sav (SEQ ID NO 2) encoded by the gene sav consists of 128 amino acids. It has a theoretical molecular weight of 15.3 kDa and an estimated pI of 4.62. Homology searches revealed homology to a number of putative proteins in related phages of the 936 and c2 species. However, no homology was found to proteins with known function. Nor was found any conserved domains in the protein. The protein is thus new and it has not previously been associated with sensitivity to phage resistance mechanisms. SEQ ID NO 7 is present in the database under accession number NC001706 and it has not previously been associated with sensitivity to phage resistance mechanisms.

The interaction of Sav with AbiV is not known but the insensitivity to AbiV of phages with a deleted sav gene clearly indicates that sav is involved in sensitivity of the phage to the φrm.

Co-expression of abiV and sav in host cells will most likely enhance the efficiency of AbiV since the escaping mutant phages will have to mutate in other genes than sav. Co-expression might also broaden the range of phages against which AbiV is effective. These are so far only hypotheses but they are in the process of being tested experimentally.

Since sav has not previously been associated with any φrm, the AbiV φrm in the present invention is a new abi mechanism interacting in a so far unknown way with the sensitive phage. AbiV is therefore likely to be an efficient φrm capable of supplementing already isolated and used phage resistance mechanisms thus improving the field of phage resistance mechanisms.

The discovery of a phage gene involved in sensitivity to the Abi-resistance mechanism may be used for obtaining a phage resistance mechanism that is more efficient than use of the AbiV-mechanism alone. It is thus likely that the use of the wild type orf26-sequence encoding the polypeptide according to SEQ ID NO 2 and/or SEQ ID NO 7 will fully or partly prevent that the phage can escape the Abi-mechanism according to the present invention by supplying AbiV-sensitive protein (SaV) together with AbiV-protein.

The present invention thus relates to the use of polynucleotide sequences encoding both SEQ ID NO 1 and SEQ ID NO 2 and/or SEQ ID NO 7 (or a variant thereof) within a Lactococcus cell in order to exploit the synergy that exists in this combination. Compared to other known Abi-systems, the combination of SEQ ID NO 1 and SEQ ID NO 2 and/or SEQ ID NO 7 (or a variant thereof) in the same cell provides for a phage resistance mechanism that is extraordinarily efficient in preventing phage infections and thus preventing the emergence of AbiV-resistant phages.

Example 16

Use of Bacteria According to the Invention

The φrm according to the present invention can be used in connection with dairy starter cultures in existing dairy production plants to produce any fermented dairy food product.

Example 17

Construction of Expression Vector pLC5

The pGKV259 vector (18) was used as the starting molecule from which pLC5 was derived. pGKV259 was digested with PstI (located downstream from the P59 promoter) followed by gel purification. Two complementary oligonucleotides (5′-TGGATCCAAAGGAGGTCCTGCA-3′ and 5′-GGACCTCCTTTGGATCCATGCA-3′) were annealed together using standard procedures (16) to create a double stranded linker with PstI-compatible sticky ends. This linker also contained a unique BamHI site and a ribosome binding site (RBS: 5′-AGGAGG-3′). The linker was inserted into the PstI site of pGKV259 and the ligation mixture was transformed into E. coli MC1061. Transformants were selected on LB plates containing 20 μg/ml chloramphenicol. Positive clones with the linker inserted in the right direction were identified by colony PCR. Correct clones were later confirmed by sequencing. Upon introduction of the linker into pGKV259, the PstI site on the 5′-side of the linker was disrupted whereas the one on the 3′-side was conserved. Thus, a unique PstI site was created 8-bp downstream from the RBS. Cloning of an insert harboring its own ATG start codon into the PstI site of pLC5 enables efficient transcription from the P59 promoter, and translation from the introduced RBS. For this invention, however, the native RBS of AbiV and not the RBS in the vector was used for translation of the protein.

Example 18

RNA Isolation, Purification and RT-PCR Analysis of Transcription

Overnight cultures were diluted 100-fold and grown to OD₆₀₀=0.5 at 37° C. Aliquots (2 ml) were harvested by quick centrifugation (20,000 g, 30 sec) and the pellet was resuspended in a solution of 0.5 M sucrose with 60 mg/ml lysozyme. Following incubation (37° C., 15 min), the cells were pelleted and resuspended in 1 ml TRIzol Reagent (Invitrogen). Total RNA was isolated according to the manufacturer's instructions. Prior to reverse transcription (RT)-PCR, the RNA samples were treated with the DNase based TURBO DNA-free kit (Applied Biosystems).

RT-PCR was carried out using the RevertAid First Strand cDNA Synthesis kit (Fermentas) as recommended by the manufacturer. As a control, the RT-PCR procedure was carried out without reverse transcriptase to ensure that the RNA samples were free of contaminating DNA.

Example 19

Mutants of L. lactis subsp. cremoris MB112 Spontaneously Expressing AbiV

Cultures of L. lactis MB112 in exponential growth were mixed with the lytic phage sk1 (MOI>1) in presence of 10 mM CaCl₂ and incubated 10 min at room temperature before plating and incubation at 36° C. overnight. Spontaneous mutants were observed with a frequency of ca. 10⁻⁸. Forty single colonies were purified and cross-streaked against phages sk1, p2, 712 and p2.1. A bacterial mutant expressing AbiV is expected to be resistant to sk1 and p2 but sensitive to 712 and p2.1 (Table 1 and Table 2). Possible candidates were tested with EOP for resistance to phages p2, 712, p2.1. One mutant (JH-80) revealed the expected pattern of a mutant expressing AbiV with EOP values of 2×10⁻⁵, 0.75 and 0.8, respectively.

This mutant was investigated for transcription of the abiV gene using reverse transcriptase PCR (RT-PCR) (FIG. 6), as described in example 18.

These results demonstrate that it is possible to obtain mutants of L. lactis strains carrying abiV on the chromosome which spontaneously express AbiV. This experiment demonstrates that it is possible to obtain phage resistant bacteria expressing AbiV without using genetic modification. This is particular interesting for the dairy industry that prefers to avoid the use of genetically modified organisms (GMO).

Example 20

Conjugal Transfer of abiV

In order to improve the non-GMO alternative of the φrm invention a conjugation experiment was conducted in which the φrm was transferred from the chromosome of JH-32 (donor) to MG1614 (recipient) (Table 4). Briefly, donor and recipient were recovered from plates and mixed at high cell densities (OD₆₀₀=40). After 2 min incubation the cell mixture was plated on non-selective plates and incubated overnight in anaerobic conditions. The cells were then recovered from the plates and plated with selection for donor (erythromycin resistance), recipient (rifampicin resistance) and transconjugants (erythromycin and rifampicin resistance), respectively.

Since the erythromycin resistance gene is inserted just upstream of abiV in JH-32 the erythromycin resistance phenotype was used to select for transfer of this gene to MG1614 hoping that abiV would be transferred along with it. Rifampicin resistance was used to select for MG1614.

A number of transconjugant candidates were isolated and purified. The additional phenotypes (resistance to phage, streptomycin and fluorouracil) were used to test the isolated candidates for verification of the phage+erythromycin resistance from JH-32 to MG1614. In JH-81 the expected pattern was observed (Table 4). An EOP value of 10⁻⁴ which is similar to other EOP values obtained with AbiV (Table 2) makes it plausible that abiV was transferred and expressed in MG1614.

This experiment demonstrated that it is possible to transfer AbiV by conjugation from one bacterium to another. Conjugation is not considered as genetic modification and the method is thus suitable for the industry for transferring AbiV between bacterial strains in a non-GMO manner.

TABLE 4 Phenotype of donor (D), recipient (R) and transconjugant (T). Selection for transconjugants was done using erythromycin and rifampicin. Erythro- Strepto- Resistance Phage mycin Rifampicin mycin Fluorouracil JH-32 (D) yes yes no no yes MG1614 (R) no no yes yes no JH-81 (T) yes yes yes yes no References in Table 1

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The invention claimed is:
 1. A starter culture composition comprising a Lactococcus lactis bacterium comprising a recombinant expression vector, wherein said vector encodes a polypeptide with at least 70% identity with SEQ ID NO 1 or a fragment thereof with a size of least 100 amino acids, wherein expression of said polypeptide confers at least one phage resistance mechanism to said Lactococcus lactis bacterium.
 2. The starter culture composition according to claim 1, wherein said Lactococcus lactis bacterium further comprises a recombinant expression vector comprising a polynucleotide encoding a polypeptide with at least 70% identity with a sequence selected from the group consisting of: SEQ ID NO 2, a fragment of SEQ ID NO 2 that is at least 100 amino acids, SEQ ID NO 7, and a fragment of SEQ ID NO 7 that is at least 100 amino acids.
 3. A method of improving phage resistance in a Lactococcus lactis bacterium comprising providing a Lactococcus lactis bacterium with an isolated polynucleotide sequence that encodes a polypeptide with at least 70% identity with SEQ ID NO 1 or a fragment thereof with a size of least 100 amino acids.
 4. The method of claim 3, further comprising providing said Lactococcus lactis bacterium with an isolated polynucleotide that encodes a polypeptide with at least 70% identity with a sequence selected from the group consisting of SEQ ID NO 2, a fragment of SEQ ID NO 2 that is at least 100 amino acids, SEQ ID NO 7, and a fragment of SEQ ID NO 7 that is at least 100 amino acids.
 5. A method of making a fermented food product comprising adding the starter culture according to claim 1 to a food product prior to fermentation of said food product, wherein said food product is selected from the group consisting of sour cream, creme fraiche, buttermilk, butter, cheese, cottage cheese, quark, cream cheese, fromage frais, yoghurt, fruit juices, fermented vegetables/fruits, and processed meat products.
 6. A method of making a fermented food product comprising adding the starter culture according to claim 2 to a food product prior to fermentation of said food product, wherein said food product is selected from the group consisting of sour cream, creme fraiche, buttermilk, butter, cheese, cottage cheese, quark, cream cheese, fromage frais, yoghurt, fruit juices, fermented vegetables/fruits, and processed meat products.
 7. A phage resistant Lactococcus lactis bacterium comprising pGhost9::ISS1 and a polynucleotide sequence encoding SEQ ID NO
 1. 8. The starter culture composition according to claim 1, wherein said Lactococcus lactis bacterium comprises a recombinant expression vector that encodes a polypeptide with at least 70% identity with SEQ ID NO 1 and, wherein expression of said polypeptide confers at least one phage resistance mechanism to said Lactococcus lactis bacterium.
 9. The starter culture composition according to claim 1, wherein said Lactococcus lactis bacterium comprises a recombinant expression vector that encodes a polypeptide with at least 80% identity with SEQ ID NO 1 and, wherein expression of said polypeptide confers at least one phage resistance mechanism to said Lactococcus lactis bacterium.
 10. The starter culture composition according to claim 1, wherein said Lactococcus lactis bacterium comprises a recombinant expression vector that encodes a polypeptide with at least 90% identity with SEQ ID NO 1 and, wherein expression of said polypeptide confers at least one phage resistance mechanism to said Lactococcus lactis bacterium.
 11. The starter culture composition according to claim 1, wherein said Lactococcus lactis bacterium comprises a recombinant expression vector that encodes a polypeptide having the sequence of SEQ ID NO 1 and, wherein expression of said polypeptide confers at least one phage resistance mechanism to said Lactococcus lactis bacterium.
 12. The starter culture composition according to claim 11, wherein said Lactococcus lactis bacterium further comprises a recombinant expression vector comprising a polynucleotide encoding a polypeptide with at least 70% identity with a sequence selected from the group consisting of: SEQ ID NO 2, a fragment of SEQ ID NO 2 that is at least 100 amino acids, SEQ ID NO 7, and a fragment of SEQ ID NO 7 that is at least 100 amino acids.
 13. The starter culture composition according to claim 8, wherein said Lactococcus lactis bacterium further comprises a recombinant expression vector comprising a polynucleotide encoding a polypeptide with at least 70% identity with a sequence selected from the group consisting of: SEQ ID NO 2 and SEQ ID NO
 7. 14. The starter culture composition according to claim 9, wherein said Lactococcus lactis bacterium further comprises a recombinant expression vector comprising a polynucleotide encoding a polypeptide with at least 80% identity with a sequence selected from the group consisting of: SEQ ID NO 2 and SEQ ID NO
 7. 15. The starter culture composition according to claim 10, wherein said Lactococcus lactis bacterium further comprises a recombinant expression vector comprising a polynucleotide encoding a polypeptide with at least 95% identity with a sequence selected from the group consisting of: SEQ ID NO 2 and SEQ ID NO
 7. 16. The starter culture composition according to claim 11, wherein said Lactococcus lactis bacterium further comprises a recombinant expression vector comprising a polynucleotide encoding a polypeptide having a sequence selected from the group consisting of: SEQ ID NO 2 and SEQ ID NO
 7. 17. The starter culture composition according to claim 11, wherein said Lactococcus lactis bacterium further comprises a recombinant expression vector comprising a polynucleotide encoding a polypeptide having the sequences of SEQ ID NO 2 and SEQ ID NO
 7. 18. A composition comprising a Lactococcus lactis bacterium that comprises a recombinant expression vector, wherein said vector encodes a polypeptide with at least 70% identity with SEQ ID NO 1 or a fragment thereof with a size of least 100 amino acids, wherein expression of said polypeptide confers at least one phage resistance mechanism to said Lactococcus lactis bacterium.
 19. The composition according to claim 18, wherein said recombinant expression vector comprises a sequence that encodes a polypeptide with at least 70% identity with SEQ ID NO 1 and, wherein expression of said polypeptide confers at least one phage resistance mechanism to said Lactococcus lactis bacterium.
 20. The composition according to claim 18, wherein said recombinant expression vector comprises a sequence that encodes a polypeptide having the sequence of SEQ ID NO 1 and, wherein expression of said polypeptide confers at least one phage resistance mechanism to said Lactococcus lactis bacterium.
 21. The composition according to claim 18, wherein said Lactococcus lactis bacterium further comprises a recombinant expression vector that comprises a polynucleotide encoding a polypeptide with at least 70% identity with a sequence selected from the group consisting of: SEQ ID NO 2, a fragment of SEQ ID NO 2 that is at least 100 amino acids, SEQ ID NO 7, and a fragment of SEQ ID NO 7 that is at least 100 amino acids.
 22. The composition according to claim 21, wherein said Lactococcus lactis bacterium further comprises a recombinant expression vector that comprises a polynucleotide encoding a polypeptide with at least 70% identity with a sequence selected from the group consisting of: SEQ ID NO 2 and SEQ ID NO
 7. 23. The composition according to claim 21, wherein said Lactococcus lactis bacterium further comprises a recombinant expression vector that comprises a polynucleotide encoding a polypeptide having a sequence selected from the group consisting of: SEQ ID NO 2 and SEQ ID NO
 7. 24. The composition according to claim 21, wherein said Lactococcus lactis bacterium further comprises a recombinant expression vector that comprises a polynucleotide encoding a polypeptide having the sequences of SEQ ID NO 2 and SEQ ID NO
 7. 25. The composition of claim 18, wherein said composition is a food product selected from the group consisting of sour cream, creme fraiche, buttermilk, butter, cheese, cottage cheese, quark, cream cheese, fromage frais, yoghurt, fruit juices, fermented vegetables/fruits, and processed meat products.
 26. The composition of claim 21, wherein said composition is a food product selected from the group consisting of sour cream, creme fraiche, buttermilk, butter, cheese, cottage cheese, quark, cream cheese, fromage frais, yoghurt, fruit juices, fermented vegetables/fruits, and processed meat products. 